Diarylation of thiazolopyrimidines by laccase and their in vitro evaluation as antitumor agents

A mild and efficient method was developed for the synthesis of new derivatives of thiazolo[3,2-a] pyrimidin-3(2H)-ones from available starting materials based on the oxidation of catechols to ortho-quinone by Myceliophthora thermophila laccase (Novozym 51,003) and 1,4-addition of active methylene carbon to these in situ generated intermediates in moderate to good yields (35–93%). The structure of the products was confirmed through 1H NMR, 13C NMR, HMBC, HSQC, DEPT-135, and mass spectroscopy techniques. These novel compounds were evaluated as active antitumor agents against human colorectal adenocarcinoma and liver adenocarcinoma cell lines. All compounds displayed potent inhibition activities against the HT-29 cell line with IC50 values of 9.8–35.9 µM, superior to the positive control doxorubicin, and most showed potent anticancer activities against the HepG2 cell line.


Result and discussion
Here we report a new strategy for the synthesis of gem-dicatechols through laccase-catalyzed oxidation of catechol to ortho-quinone and subsequent nucleophilic attack of active methylene carbon. First, 0.50 mmol of 6-acetyl-7-methyl-5-phenyl-2H-thiazolo[3,2-a] pyrimidin-3(5H)-one (2a) and 1 mmol of catechol (3a) as a model reaction were reacted in a mixture of phosphate buffer (0.01 M, pH 8) and CH 3 CN (2:1 v/v) at room  www.nature.com/scientificreports/ temperature using a commercially available laccase (Novozym 51,003). The progress of the reaction was detected with TLC for 24 h. it turned out that the best reaction time was 4 h because the yield of product did not change after that. After workup with ethyl acetate and purification with silica gel column chromatography, product 4a obtained in moderately good yield (65%) ( Table 1 entry 2). As can be seen from Table 1, the model reaction was carried out in different buffer systems, such as citrate pH 3.5 and phosphate pH 4.5 and 7 with acetonitrile as a co-solvent. Reactions at pH 3.5 and 4.5 did not result in product and at pH 7, only 8% product was obtained. Also, different co-solvents, such as ethanol and methanol, were tested, but this did not result in product formation. Hence, the best condition was phosphate buffer pH 8 with acetonitrile. No product was observed when the reaction was tested without laccase (entry 4). The amount of enzyme was also optimized and the best yield was obtained when 1 ml of the commercial enzyme formulation was used for 0.1 mmol of substrate. Laccase activity was measured at various pH (4.0-8.0). It was found that the best laccase activity was at pH 4.0-5.0 (Fig. 3). But the desired reaction could not be done at these pH values. This can be due to the lack of activation of the active methylene carbon at C-2 in acidic pH to attack the ortho-quinone.
For accurate characterization of the isolated products, various identification methods were used that included 1 H NMR, 13 C NMR, H, H-COSY, HMBC, HSQC, DEPT-135 and mass spectroscopy. The DEPT-135 and HSQC spectrum (supporting information) of 4a exhibited the existence of a quaternary carbon (δ c 66.94 ppm) representing the carbon attached to two catechol molecules. Based on the HMBC spectrum of 4a (supporting information) the correlation between the quaternary carbon (δ c 66.94 ppm) and the catechol protons is well illustrated (Fig. 4). www.nature.com/scientificreports/ Also, H, H-COSY spectrum of 4a revealed the presence of three spin systems as follows: one between protons of a catechol ring at δ H 6.09-6.12 (m, 1H) that overlapped with H A (Fig. 4) and two doublets at δ H 6.44 (d, J = 2.4 Hz, 1H) and δ H 6.47 (d, J = 8.4 Hz, 1H), while the second spin system occurred between protons of another catechol ring at δ H 6.69 (dd, J = 8.3, 2.5 Hz, 1H) and δ H 6.75 (m, 2H). The third spin system δ H 7.32 (m, 5H) is related to the phenyl ring B.
To explore further reaction diversity on the catechol side, 3-methyl catechol 3b was used. As shown in Table 2, this resulted in product formation with excellent yields, even better than with catechol 3a (compare 4o (93%) vs 4e (79%) and 4n (83%) vs. 4 k (68%)). When 3-methyl catechol 3b was used, it was expected that a mixture of products resulting from the nucleophilic attack on two sites (carbon 2 and 3 in Fig. 5) would be observed. However, according to the coupling constant (J = 2.4 Hz), which is caused by meta-coupling, just one product was observed that resulted from a nucleophilic attack on carbon 2.
Based on previous studies and control experiments, a possible mechanism can be proposed. It is assumed that the reaction starts with the laccase-catalyzed oxidation of catechol (3a) to o-benzoquinone (5a) (Fig. 6). This is followed by a 1,4-addition of the active methylene carbon and the formation of the intermediate (6a). Oxidation of the second catechol to o-benzoquinone and nucleophilic attack of the intermediate 6a produces the final product 4a.
Some features of this method are consistent with the principles of green chemistry 29 : this reaction was carried out in a phosphate buffer as a solvent and acetonitrile as a co-solvent with a ratio of 2:1, which is considered a safe environment. Also, in terms of energy efficiency, this reaction is done at room temperature. In this method, there is no need to protect functional groups and therefore reduce derivatives.
To evaluate the antitumor potency of prepared compounds (4a-d,4f.-k and 4o), their in vitro cytotoxic activity was evaluated against two different cancer cell lines HT-29 (human colorectal adenocarcinoma) and HepG2 (liver adenocarcinoma) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 30 . HT-29 and HepG2 were treated with varying concentrations of the prepared compounds for 48 h. Doxorubicin was used as a positive control (IC 50 = 63 ± 2.97 μM for HT-29 and 69 ± 3.71 μM for HepG2 at 48 h). The antiproliferative efficacy data are presented as IC 50 values defined as the concentration of the compound that decreases cell proliferation at 50%. The IC 50 values were generated from at least three independent experiments.
Besides, it was worth mentioning that all target compounds except 4c and 4 k showed potent anticancer activities against the HepG2 cell line and were more potent than the positive control doxorubicin. It is noteworthy that 4o showed excellent results with both HT-29 and HepG2.

Conclusions
To summarize, a simple and efficient method was developed to synthesize new derivatives of thiazolopyrimidin-3(2H)-ones through oxidation of catechols by laccase to related o-quinones and nucleophilic attack of active methylene carbon of thiazolopyrimidin-3(2H)-ones to these intermediates. The reaction was performed at room temperature and used aerial oxygen as an oxidant. These novel compounds were evaluated as active antitumor  www.nature.com/scientificreports/ www.nature.com/scientificreports/ agents against human colorectal adenocarcinoma and liver adenocarcinoma cell lines. All compounds displayed potent inhibition activities against the HT-29 cell line with IC 50 values of 9.8-35.9 µM, superior to the positive control doxurobicin, and most showed potent anticancer activities against the HepG2 cell line. Future mechanistic studies are unambiguously needed to elucidate anticancer effects of our compounds, providing an improved basis for its prediction in further clinical studies.   Determination of the optimum pH activity:. 100 µl of laccase solution was added to 2 ml of buffers at different pH levels (4.0-8.0) and incubated at 25 °C and 200 rpm for 24 h. the activities of samples were measured at the same pH levels.
The activity of laccase. The activity was measured spectrophotometrically (Thermo Electron, model UV1 spectrophotometer) with ABTS as substrate (1 mM) in 100 mM sodium citrate buffer pH 4.5. To measure the laccase activity, 5 μl of enzyme solution was added to 965 μl of related buffer and 30 μl of the ABTS (1 mM) solution at room temperature to start the oxidation reaction. The change in absorbance at 420 nm (ε = 36 × 10 3 M −1 cm −1 ) was recorded for 40 s (per 5 s) and the catalytic activity was assayed by calculating the slope of the initial linear segment of the kinetic curve 31 . One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of ABTS per min and the activities were expressed in U/g.

Synthesis of dihydropyrimidines (DHPMs).
According to the literature procedure 32 , DHPMs 1a-1i and 1j-1 m were prepared by the following method. A mixture of benzaldehyde derivatives (5 mmol), acetylacetone or ethyl acetoacetate (5 mmol), thiourea (7.5 mmol), and NH 4 Cl (0.8 mmol) was heated with stirring at 100 °C for 3 h. After cooling, the reaction mixture was washed with cold water and the residue recrystallized from ethanol. DHPMs 1f.-1 h were prepared by a mixture of benzaldehyde derivatives (5 mmol), dimedone (5 mmol), thiourea (7.5 mmol) and a few drop H 2 SO 4 in ethanol (15 ml) was heated under reflux overnight. After cooling, the reaction mixture was dropped in cold water and the precipitated residue recrystallized from ethanol.

Synthesis of thiazolopyrimidin-3(2H)-ones (2a-m).
According to the literature procedure 28 a mixture of corresponding DHPMs (1 mmol) and ethyl chloroacetate (1 ml, 90 mmol) was heated for 30 min at 110-115° C. The solution was cooled, and the precipitate was filtered off and washed with EtOAc. The prepared hydrochloride was dissolved in EtOH (10 mL) and ammonia was added to adjust pH 7.5-8.0. Evaporation of the solvent at the reduced pressure, followed evaporation from the chloroform solution gave pure compounds 2a-m. Typical experimental procedure for the synthesis of product 4. A 100 mL round bottom flask with a magnetic stir bar was charged with a solution or suspension of a catechol 3 (1 mmol) and a 2 (0.50 mmol) in acetonitrille (8 mL). Phosphate buffer (0.10 mM, pH 8.0, 16 mL) and Myceliophthora thermophila laccase (Novozyme 51,003) (5 ml, 2.47 g, 5000 U) were added and the mixture was stirred under air. The reaction progress was detected with thin-layer chromatography until thiazolo[3,2-a] pyrimidine was completely consumed (4 h). Then the reaction mixture was diluted with EtOAc. The layers were decanted and the aqueous phase was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine, dried over anhydrous Na 2 SO 4 and filtered, and removed the solvents in vacuo. Purification by preparative thin layer chromatography (Hexane: EtOAc) (3:1) provided target compound 4.

6-acetyl-7-methyl-5-(3-nitrophenyl)-2H-thiazolo[3,2-a]pyrimidin
MTT assay. The prepared compounds (4a-d,4f.-k and 4o) were examined to assess their cytotoxicity effects against two cancer cell lines (human colorectal adenocarcinoma cell line HT-29 and liver adenocarcinoma cell line HepG2) using a standard MTT-based colorimetric assay. These cells were cultured in RPMI 1640 medium supplemented with 10% heat-activated fetal bovine serum (FBS). A day before treatment, appropriate numbers of cells were seeded into each well of 96-well plates (Corning, New York, USA) and incubated at 37 °C with 5% CO 2 . Testing compounds at pre-set concentrations were added to each well with doxorubicin being used as a positive control. Each concentration was in triplicate. After 48 h exposure period, MTT reagent was added (0.5 mg/mL) to the wells and incubated for the next 1 to 2 h at 37 °C, then the medium was replaced by DMSO to www.nature.com/scientificreports/ dissolve the purple formazan crystals formed. The optical absorbance of each well was determined by an ELISA plate reader at 570 nm.

Data availability
All data generated or analysed during this study are included in this published article and its supplementary information files.